This invention relates to an improved test for assessing the unsaturated binding capacity of serum proteins which bind thyroid hormones and is more particularly concerned with the use of an improved sorbent in such a test.
The thyroid gland synthesizes triiodothyronine (T3) and thyroxine (T4) and releases them into the circulation, where approximately 99.9 percent of these hormones are bound to serum proteins and the remaining small fractions of the thyroid hormones (T3 and T4) circulate in free form. Due to the influence that T3 and T4 have on the metabolic processes, these naturally occuring iodinated amino acids have been the subject of many investigations by thyroid physiologists. From these investigations, the protein components in blood serum which bind the thyroid hormones have been identified and characterized.
Further studies have shown that various diseases, genetic abnormalities, and drugs can alter the concentrations or affinities of the various binding proteins, which sometimes will cause a concurrent rise or fall in the free or unbound hormone level. Due to the profound catabolic effects that the free thyroid hormones exhibit, an assessment of the binding capacity of the serum proteins is often routinely performed as part of a thyroid profile.
At least three serum protein fractions which have different affinities and capacities for thyroxine have been identified by reverse flow paper electrophoresis. Thyroxine binding globulin (TBG) has the highest affinity for T4 and binds 65 to 75% of circulating hormone; albumin has a low affinity and binds only about 10% of the hormone; and thyroxine binding prealbumin (TBPA) has an intermediate affinity for T4 and binds about 15 to 25% of the hormone.
It has been shown that triiodothyronine binds less firmly but in different degrees to the same thyroxine binding proteins. Approximately 70% of the T3 is bound to TBG; 30% is bound to albumin; and a non-detectable amount to TBPA.
The metabolic processes are regulated entirely by the small amounts of free T3 and T4 in circulation. Therefore, the best measure of the thyrometabolic status would be a free hormone level. Presently, the procedures which are available for assessing the levels of free thyroid hormones are very time-consuming and cumbersome to perform. As early as 1965, Clark and Horne demonstrated an indirect method for the estimation of free T4 levels by using the total T4 level and the level of unsaturated binding proteins (T3 Uptake).
A T3 Uptake test is a qualitative assessement of the unsaturated binding capacity of the serum proteins which bind the thyroid hormones. In hypothyroidism, the thyroid hormone levels are low, leaving the binding proteins relatively unsaturated. While in hyperthyroidism, the thyroid hormone levels are elevated, causing a high degree of saturation of the binding proteins.
The first attempt to measure the unsaturated binding capacity was described by Hamolsky in 1957. The test was performed by adding radioactive T3 to a sample of whole blood and measuring the uptake of labelled T3 by the red blood cells. The Hamolsky red blood cell uptake test provided an important in vitro clinical tool, but it had severe limitations caused by variations in the washing technique.
Mitchell taught the use of an ion exchange resin and resin sponge to bind the radioactive T3 not bound to the serum proteins. This method of separation suffered from time and temperature dependence. Since then, various other adsorbents such as coated charcoal, silicates, Sephadex.RTM., and organic polymers have been utilized as separating agents in T3 uptake tests, all of which have disadvantages.
The following is a list of desirable characteristics of a sorbent for use in a T3 uptake or similar test:
1. Sorbent reaction should not be temperature dependent over a temperature range usually encountered in laboratories. PA1 2. Time dependence should allow the sorbent reaction to reach equilibrium in 15 to 20 minutes and remain stable for at least 60 minutes. PA1 3. Addition of the sorbent should be capable of being carried out by pipetting a suspension of the sorbent or by inserting a sorbent tablet. Ideally, the material should remain in suspension for 30 minutes or longer without any further mixing, or be easily tableted. PA1 4. The sorbent should be stable in suspension or tablet form for at least four months or longer. PA1 5. The sorbent should be easily separated from the reaction mixture by usual laboratory methods, such as centrifugation at 1500 g or less and decantation. PA1 6. The sorbent should not precipitate serum proteins, since both the free and bound .sup.125 I-T3 would be adsorbed and no separation would be achieved. PA1 7. The sorbent should not be toxic or caustic. PA1 8. The sorbent should not react with glass or polystyrene, since most tests are performed in test tubes made of these materials. PA1 9. The sorbent should not be prohibitively expensive.
The sorbents employed in the present invention meet these criteria.